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The Role of the Orphan Nuclear Receptor, Liver Receptor Homologue-1, in the Regulation of Human Corpus Luteum 3␤-Hydroxysteroid Dehydrogenase Type II

NOEL PENG, JOUNG W. KIM, WILLIAM E. RAINEY, BRUCE R. CARR, AND GEORGE R. ATTIA Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas Texas 75390-9032

After ovulation, ovarian 3␤-hydroxysteroid dehydrogenase level. Cell transfection, mutation analysis, and EMSA were type II (HSD3B2) expression increases to enhance the shift of performed to examine the role of LRH-1 in the regulation of steroidogenesis toward progesterone biosynthesis. Steroido- HSD3B2. LRH-1 expression was higher in CL, compared with genic factor-1 (SF-1) is a transcription factor for several genes mature ovarian follicles. Cotransfection of granulosa cells encoding steroidogenic enzymes. However, the level of SF-1 with HSD3B2 and LRH-1 resulted in a 10-fold increase of tran- expression decreases in the human corpus luteum (CL) after scription. DAX-1 inhibited LRH-1-stimulated HSD3B2, which ovulation. Liver receptor homolog-1 (LRH-1) is another mem- was maintained in the presence of dibutyryl cAMP. Mutation ber of the orphan nuclear receptor family. We hypothesize of the either of the two putative LRH-1 binding sites, which that LRH-1, rather than SF-1, plays an essential role in the were confirmed by EMSA, in the HSD3B2 promoter decreased regulation of corpus luteum steroidogenesis. Semiquantita- LRH-1 stimulation. Our findings suggest that LRH-1 is highly tive RT-PCR and real-time PCR were performed to quantify expressed in CL, and it plays an essential role in the regulation the level of LRH-1 expression and correlate with HSD3B2 of HSD3B2. (J Clin Endocrinol Metab 88: 6020–6028, 2003)

VARIAN PRODUCTION OF progesterone and estro- Orphan nuclear receptors are transcription factors for O gen is determined by the cell-specific expression of a which ligands have not been identified. Two such factors, variety of steroidogenic enzymes and transcription factors steroidogenic factor-1 (SF-1; designated NR5A1) and dosage- (1–4). Although the tropic hormones and steroidogenic en- sensitive sex reversal-adrenal hypoplasia congenita critical zymes responsible for ovarian steroid hormone biosynthesis region on the X chromosome, gene 1 (DAX-1; designated have been well defined, the mechanisms controlling ex- NR0B1) are involved in the regulation of steroidogenesis pression of steroid-metabolizing enzymes remain an area (13–15). SF-1 has been demonstrated as a regulator of the of active research. After ovulation, there is a shift from transcription of several genes encoding steroidogenic en- a predominantly estrogen-producing ovarian follicle to a zymes including HSD3B2 (16–22). DAX-1 tissue expression predominantly progesterone-producing corpus luteum (CL). colocalizes with SF-1 and has the ability to inhibit SF-1- This shift in steroidogenesis is still poorly understood, but mediated transcription (6, 24). alterations in gene transcription appear to be a major mech- Liver receptor homolog-1 (LRH-1; designated NR5A2) is anism controlling this transition (5–8). another orphan nuclear receptor, which is expressed in mul- 3␤-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a tiple tissues (25–29). LRH-1 and SF-1 are members of the microsomal enzyme that is present in the theca interna and nuclear receptor family that recognize the canonical recog- granulosa cells (9, 10). During the follicular phase, thecal cells nition motif for Drosophila fushi tarazu factor receptors and express low levels of HSD3B2 for C19 steroid production to bind to DNA as monomers to enhance transcription of tar- occur. After ovulation, there is an increase in ovarian follicle geted genes. LRH-1 has an overall 60% amino acid similarity HSD3B2 expression that enhances the shift of steroid pro- to SF-1 with virtually identical DNA binding domain. Ini- duction toward progesterone biosynthesis. HSD3B2 mRNA tially, it was thought that LRH-1 was only expressed in levels increase greatly in the corpora lutea and particularly tissues derived from the gut endoderm such as pancreas, the luteinized granulosa cells at a time when progesterone liver, and intestine (27–29). More recently, LRH-1 was found becomes the primary ovarian product (11, 12). in mouse, equine, and human ovaries (29–31), human testis, and at low levels in the human adrenal (32), raising the Abbreviations: DAX-1, Dosage-sensitive sex reversal-adrenal hypo- possibility that LRH-1 could play a role in regulation of plasia congenita critical region on the X chromosome, gene 1; dbcAMP, steroidogenesis. However, the role of LRH-1 in the regula- dibutyryl cAMP; DME/F12, DMEM and Ham’s F-12 medium; FP, free tion of ovarian steroidogenesis is still poorly defined. probe; GCT, granulosa cell tumor; G3PDH, glyceraldehyde-3-phosphate In human ovarian cycle, the LH surge is essential for ovu- dehydrogenase; HLGC, human luteinized granulosa cell; HSD3B2, 3␤- hydroxysteroid dehydrogenase type II; IVP, in vitro protein; LRH-1, liver lation and subsequent conversion of mature ovarian follicle receptor homologue-1; NE, nuclear extract; PKA, protein kinase A; RT, to CL. Although SF-1 can regulate enzymes that are essential reverse transcription; SF-1, steroidogenic factor-1. for progesterone production, the expression of SF-1 was

6020 Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 6021 found to be down-regulated in ovarian cells after LH surge TABLE 1. Primers used for RT-PCR and real-time PCR analyses (33–35), which raises questions regarding the role of SF-1 in of human ovary corpus luteum steroidogenesis. Thus, we hypothesize that Transcript Primer Length LRH-1, rather than SF-1, could play an essential role in the LRH-1a 5Ј-TGAAGCTGCTTCAGAAC TGC-3Ј 448 bp regulation of human corpus luteum function and expression 5Ј-CCGTTCAGGTGC TTGTAGTA-3Ј of steroidogenic enzymes, specifically HSD3B2. SF-1a 5Ј-GCAGAAGAAGGCACAGATTC-3Ј 824 bp In this study we examined the differential expression of 5Ј-TCACCAGGATGTGGTTATTC-3Ј LRH-1 between human mature ovarian follicles and CL. We G3PDHa 5Ј-CCACCCATGGCAAATTCCATGGCA-3Ј 625 bp 5Ј-CTAGACGGCAGGTCAGGTCCACC-3Ј also examined the role of LRH-1 in the regulation of HSD3B2 LRH-1b 5Ј-TACCGACAAGTGGTACATGGAA-3Ј 89 bp promoter. 5Ј-CGGCTTGTGATGCTATTATGGA-3Ј SF-1b 5Ј-GGAGTTTGTCTGCCTCAAGTTCA-3Ј 80 bp 5Ј-CGTCTTTCACCAGGATGTGGTT-3Ј

Materials and Methods Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021 HSD3B2b 5Ј-GCGGCTAATGGGTGGAATCTA-3Ј 128 bp Cell preparation and RNA extraction 5Ј-CATTCTTGTTCAGGGCCTCAT-3Ј 5Ј-TGATACCTTGTACACTTGTGCGTTAAGACC-3Ј Ovarian cortex, ovarian follicle, and corpus luteum were isolated (probe) from human ovarian tissues. Human ovaries were obtained from women of reproductive age (aged 30–40 yr) at the time of hysterectomy a RT-PCR. and were determined to be normal. Human luteinized granulosa cells b Real-time PCR. (HLGCs) were obtained by follicular aspiration from women of reproductive age (aged 25–35 yr) undergoing oocyte retrieval for in vitro fertilization. Briefly, women were treated with GnRH agonist before and contained water instead of first-strand cDNA. Each sample was nor- during follicular stimulation using recombinant human gonadotropin. malized on the basis of its 18S rRNA content. The 18S quantification was After follicular aspiration, HLGCs were isolated as previously described performed using the TaqMan rRNA control reagent kit (Applied Bio- (36). The HLGCs were washed twice with DMEM and Ham’s F-12 systems) and following the method protocol provided by the medium (DME/F12) (GIBCO BRL, Gaithersburg, MD) and then incu- manufacturer. bated for 30 min at 37 C in DME/F-12 containing 0.1% hyaluronidase to disperse them. The dispersed cells were resuspended in 20 ml me- Preparation of reporter constructs and expression vectors dium and transferred to 50-ml tubes containing 3.5 ml Histopaque 1077 The 5Ј flanking DNA from the human HSD3B2 gene(Ϫ963 bp) (22) (Sigma Chemical Co., St. Louis, MO). HLGCs were separated from red was inserted upstream of the firefly luciferase gene in the reporter vector blood cells by centrifugation at 600 ϫ g for 15 min. HLGCs formed a thin pGL3-Basic (Promega, Madison, WI). For all transfections, empty pGL3- layer between the Histopaque and the medium. Cells were removed and Basic was used as the control vector to measure basal activity. The coding then washed three times using DME/F-12 containing 5% fetal bovine region of LRH-1 (provided by Dr. David Mangelsdorf, University of serum; 5% horse serum; 1% ITS Plus (Collaborative Research, Waltham, Texas Southwestern) and SF-1 (39) were inserted into pcDNA3 (Invitro- MA); 2% Ultroser G (IBF Biotechnics, Sepracor, Inc., Marlborough, MA); gen) eukaryotic expression vector. and antibiotics. Total RNA was extracted from ovarian cortex, mature ovarian follicles, CL, and HLGCs as previously described (37). Purity and integrity Cell culture and transfection assay of RNA was checked spectroscopically and by gel electrophoresis before Human ovarian granulosa cell model comprised of granulosa cell use. The use of human tissues was approved by the Institutional Review tumor (GCT) cells isolated from patient with GCT as previously de- Board of the University of Texas Southwestern Medical Center at Dallas. scribed (40). This model is able to reproduce many of the differentiated functions of granulosa cells. Specifically, they have maintained the pro- RT-PCR duction of progesterone and are able to convert androstenedione to estradiol. This cell model also responds to forskolin and dibutyryl cAMP Total RNA (1 ␮g) prepared from human ovarian follicles, CL, and (dbcAMP) by increasing production of progesterone and estradiol (data ovarian cortex was used for reverse transcription (RT) reaction in a final not shown). GCT cells were cultured in DME/F12 (GIBCO BRL) sup- volume of 20 ␮l. PCR was performed using equal amounts of RT product plemented with 5% NU Serum (Collaborative Biomedical, Bedford, MA) standarized to the amounts of glyceraldehyde-3-phosphate dehydroge- and antibiotics. For transfection experiments, Fugene 6 (Roche, India- nase (G3PDH) present as template with human LRH-1-specific primers napolis, IN) was used to transfect 0.5 ␮g of reporter plasmid and the (32), human SF-1-specific primers (38), and G3PDH-specific primers indicated amounts of expression vectors. pcDNA3 empty vector was (Accession AF261085) (Table 1). The PCR conditions were 94 C for 20 sec, used to assure constant amounts of DNA per well for each transfection. 54 C for 20 sec, and 72 C for 40 sec. PCR product (10 ␮l) of each sample Cells were assayed for reporter activity using the Luciferase assay sys- was loaded and electrophoresed on 1% agarose gel. tem (Promega,) 18–20 h after transfection.

RT and real-time PCR Preparation of deletions and mutant HSD3B2 5Ј-flanking constructs Four micrograms of total RNA each from human ovarian cortex, ovarian follicle, CL, and HLGCs was reverse transcribed in a final Several 5Ј-deletion plasmids were constructed using available re- volume of 100 ␮l using a high-capacity cDNA archive kit (Applied striction endonuclease sites. Mutant constructs containing mutated pu- Biosystem, Foster City, CA). Primers for the amplifications (Table 1) tative LRH-1 binding sites in the HSD3B2 promoter regions were gen- were based on published sequences for LRH-1 (GenBank accession no. erated by using introduction of restriction endonuclease site (EcoRI) by NM_003822), HSD3B2 (accession no. M77144), and SF-1 (NM_004959). PCR and introduction of point mutation by PCR (Table 2). PCRs were performed in the ABI Prism 7000 sequence detection system (Applied Biosystems) in a total volume of 30 ␮l reaction mixture fol- EMSA lowing the manufacturer’s recommendations using the SYBR Green Universal PCR Master Mix 2X for LRH-1 and SF-1 and Taqman 2X PCR Human ovarian GCT cell and HLGC nuclear extracts (NEs) were Master-Mix for HSD3B2 (Applied Biosystems). Forward and reverse prepared as previously described (41). The human LRH-1 was synthe- primers were added at 0.1 ␮m using the manufacturer’s recommended sized by coupled in vitro transcription/translation by T7 polymerase dissociation protocol. Standard curves were prepared using the human using the reticulocyte lysate system (Promega). Double-stranded oligo- LRH-1 (in Topo pCRII), HSD3B2 (in pVL), and SF-1 (in pcDNA3) (In- nucleotides (25 pmol) containing the wild-type and mutant LRH-1 re- vitrogen, Carlsbad, CA) eukaryotic expression vector. Negative controls sponse elements (Table 2) were labeled with [␣-32P] dCTP by Moloney 6022 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2

TABLE 2. Oligonucleotide sequences used as primers for site-specific mutagenesis or as probes for EMSA

LRH-1 site Forward Reverse 1a 5Ј-ACTGTCAGAATTCATTGACA-3Ј 5Ј-TGTCAATGAATTCTGACAGT-3Ј 5Ј-TGACAGTCTTAAGTAACTGT-3Ј 5Ј-ACAGTTACTTAAGACTGTCA-3Ј 2a 5Ј-TGGATTTACTGTACATTGACAACA-3Ј 5Ј-TGTTGTCAATGTACAGTAAATCCA-3Ј 1b 5Ј-TAACTGTCAAGGTTCATTGA-3Ј 5Ј-TCAATGAACCTTGACAGTTA-3Ј 2b 5Ј-TACTGTACAAGGACAACATT-3Ј 5Ј-AATGTTGTCCTTGTACAGTA-3Ј 1c 5Ј-TAACTGTCAgaaTTCATTGA-3Ј 5Ј-TCAATGAAttcTGACAGTTA-3Ј 2c 5Ј-TACTGTACAttGACAACATT-3Ј 5Ј-AATGTTGTCaaTGTACAGTA-3Ј a Site-specific mutagenesis. b Wild-type probe for EMSA. c Probe with mutated bases (lowercase letters) for EMSA. Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021

FIG. 1. Expression of LRH-1 and SF-1 in human ovarian tissue. Semiquantitative RT- PCR was used to measure expression of LRH-1 and SF-1. Total RNAs were extracted from human mature ovarian follicle, corpus luteum, and ovarian cortex. PCR of G3PDH was used to standardize cDNA generated from the RT reaction. At 40 cycles, we demonstrated a lower level of SF-1 expression in human corpus luteum, compared with mature ovarian follicles. In contrast, LRH-1 expression in human corpus luteum is higher, compared with mature ovarian follicles.

murine leukemia virus reverse transcriptase at 37 C for 30 min. The human corpus luteum, compared with mature ovarian fol- probes (50,000 cpm) were incubated with 5 ␮g of LRH-1 in vitro protein ␮ licles. In contrast, there was a higher level of LRH-1 expres- (IVP LRH-1), HLGC NEs, or human ovarian GCT NEs in 20 l reaction sion in human corpus luteum, compared with mature ovar- mixture [20 mm HEPES (pH 8.0), 80 mm KCL, 1 mm EDTA, 10% glycerol, 1mm dithiothreitol, 0.5 mg/ml BSA, and 0.075 mg/ml poly dI-dC to ian follicles. block nonspecific binding]. The reactions were incubated at room tem- Total RNA was isolated from human ovarian cortex, ovar- perature for 20 min. For competition analysis, reaction mixtures con- ian follicle, corpus luteum, and HLGCs and used to quantify taining 100-fold molar excess of nonradiolabeled oligonucleotide (cold) mRNA levels of LRH-1, SF-1, and HSD3B2 using real-time were added simultaneously with probe. The resulting DNA/protein complexes were then separated from free probe (FP) by electrophoresis PCR. Using these data, we compared expression of HSD3B2 using a 4% high-ionic-strength native polyacrylamide gel with 1 ϫ transcript with that found for LRH-1 and SF-1 (Fig. 2). These Tris-glycine running buffer. The gel was dried and visualized after data demonstrate that the amount of HSD3B2 mRNA ex- autoradiography at Ϫ70 C for 24 h. pression is highly correlated with the amount of LHR-1 mRNA expression in all ovarian tissue samples studied. In Statistical analysis addition, we demonstrated a higher expression of LRH-1 and Data were analyzed by ANOVA using STATPAC software (Minne- HSD3B2 in the corpora lutea and HLGCs, compared with apolis, MN). ovarian cortex and ovarian follicles. However, there was no correlation between the amount of HSD3B2 mRNA and SF-1 Results mRNA. Human ovarian follicle and corpus luteum express LRH-1 mRNA LRH-1 enhances transcription of HSD3B2 The first objective was to determine whether human cor- To test the hypothesis that LRH-1 could play a role in pus luteum and ovarian follicle expressed LRH-1 and com- progesterone biosynthesis by enhancing transcription of pare the level of expression to that seen for SF-1. To accom- HSD3B2 gene, cultured GCT cells were cotransfected with plish this goal, total RNA isolated from human ovarian HSD3B2 promoter construct alone and with expression vec- follicles, corpora lutea, and ovarian cortex were used to de- tors containing either LRH-1 or SF-1. Both LRH-1 (Fig. 3A) tect transcript levels using semiquantitative RT-PCR (Fig. 1). and SF-1 (Fig. 3B) were able to increase luciferase reporter At earlier cycles of RT-PCR, we were able to detect LRH-1 in driven by the HSD3B2 promoter in a concentration-depen- corpus luteum but not SF-1. At 40 cycles of semiquantitative dent manner. Maximal stimulation of reporter activity was RT-PCR, we showed a lower level of SF-1 expression in observed using 0.5 ␮g/well for both vectors. LRH-1 cotrans- Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 6023

FIG. 2. Quantification of LRH-1, SF-1, and HSD3B2 transcript levels in human ovarian tissues. Real-time RT-PCR was used to quantify the level of LRH-1, SF-1, and HSD3B2 mRNA in human ovarian cortex, ovarian Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021 follicle, corpora lutea, and HLGCs as described in Ma- terials and Methods. Data represent independent RNA samples and are expressed as attomoles of mRNA per microgram of 18S rRNA. Note that the data are pre- sented with a log scale. A, The amount of HSD3B2 mRNA expression highly correlated with the amount of LHR-1 mRNA expression in the corpora lutea and HLGCs. B, In contrast, there is no correlation between the amount of HSD3B2 mRNA and SF-1 mRNA.

FIG. 3. Concentration-dependent effects of LRH-1 or SF-1 on HSD3B2 reporter gene activity. GCT cells were transfected with luciferase reporter constructs containing the HSD3B2 promoter construct (0.5 ␮g/well). Cells were cotransfected with empty pcDNA3 expression vector or the indicated amounts of LRH-1 or SF-1 expression plasmid. After recovery for 24 h, cells were lysed and assayed for luciferase activity. Results represent the mean Ϯ SE of pooled data from three to four experiments. A, 0.1 (P Յ 0.05) and 0.5 (P Յ 0.01) of LRH-1 were significantly different from vector alone. B, 0.1 (P Յ 0.05), 0.3 (P Յ 0.01), and 0.5 (P Յ 0.01) of SF-1 were significantly different from vector alone. fection, however, was more effective (Ͼ8-fold) in the induc- with control (Fig. 5). In addition, the LRH-1-enhanced tion of the HSD3B2 reporter construct than SF-1 (Ͻ4-fold) HSD3B2 promoter activity was further augmented in the (Fig. 4). presence of dbcAMP. To determine whether LRH-1-enhanced HSD3B2 transcription was influenced by protein kinase A (PKA), trans- DAX-1 inhibits LRH-1 stimulated HSD3B2 transcription fected cells were treated with the cAMP analog, dbcAMP. In the absence of LRH-1, HSD3B2 reporter activity was in- Because DAX-1 can repress SF-1-mediated transcription, creased almost 3-fold after dbcAMP treatment, compared we investigated its effect on LRH-1. DAX-1 inhibited LRH-1 6024 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2 Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021

FIG. 6. Effect of DAX-1 on LRH-1-stimulated HSD3B2 promoter activity. GCT cells were transfected with luciferase reporter constructs FIG. 4. Effect of LRH-1 or SF-1 on HSD3B2 promoter activity. GCT containing the HSD3B2 promoter construct (0.5 ␮g/well). Cells were cells were transfected with luciferase reporter constructs containing cotransfected with empty pcDNA3 expression vector, LRH-1 (0.5 ␮g/ the HSD3B2 promoter construct (0.5 ␮g/well). Cells were cotrans- well), or LRH-1 plus increasing concentrations of DAX-1 expression fected with empty pcDNA3 expression vector or the indicated plasmid (␮g/well). After recovery for 24 h, cells were lysed and assayed amounts of LRH-1 (0.5 ␮g/well) or SF-1 (0.5 ␮g/well) expression plas- for luciferase activity. Results represent the mean Ϯ SE of pooled data mid. After recovery for 24 h, cells were lysed and assayed for luciferase from three to four experiments. Concentrations of DAX-1 greater than activity. Results represent the mean Ϯ SE of pooled data from three 0.03 ␮g/well were significantly different from LRH-1 alone (P Յ 0.01). to four experiments. At optimal concentrations, LRH-1 has greater effect on HSD3B2 promoter than SF-1. Both LRH-1 and SF-1 were significantly different from vector alone (P Յ 0.01). LRH-1 was sig- transcription (Fig. 7). Deletion analysis demonstrated a pu- nificantly different from SF-1 (P Յ 0.05). tative LRH-1 binding site between Ϫ963 and Ϫ515 and another between Ϫ345 and Ϫ210. Sequence analysis revealed two cis-elements similar to the nuclear receptor half-sites known to bind both LRH-1 and SF-1. Mutation of the first putative LRH-1 binding site from 5Ј-AAGGTTC-3Ј (Ϫ906/ Ϫ900) to 5Ј-AgaaTTC-3Ј decreased LRH-1 stimulation by almost half, and mutation of the second putative LRH-1 binding site from 5Ј-AAGGACA-3Ј (Ϫ315/Ϫ309) to 5Ј-Atg- GACA-3Ј completely abolished LRH-1 stimulation (Fig. 8). These inhibitions were maintained in the presence of PKA agonist (data not shown).

EMSA To confirm that LRH-1 could interact directly with the two putative response element sites, two synthetic oligonucleotides encompassing the two cis-elements were generated and FIG. 5. Effect of PKA agonist on LRH-1-stimulated HSD3B2 pro- used for EMSA (Fig. 9). IVP LRH-1, HLGC, and GCT cell NEs moter activity. GCT cells were transfected with luciferase reporter were prepared. LRH-1 bound to both sites and the protein/ constructs containing the HSD3B2 promoter construct (0.5 g/well). DNA complex was completely displaced by adding 100-fold Cells were cotransfected with empty pcDNA expression vector or LRH-1 expression plasmid. After recovery for 22 h, cells were treated molar excess of nonradiolabeled oligonucleotides (cold). In with 1 ϫ low serum (1XLS) medium or dbcAMP (1 mM). After 6 h, cells addition, when the two oligonucleotide probes were incu- were lysed and assayed for luciferase activity. Data were expressed bated with HLGC and GCT cell NEs, a similar protein/DNA as percentage of the basal. Results represent the mean Ϯ SE of pooled complex was found. These were displaced by excess nonra- data from three to four experiments. Treatment with dbcAMP was significantly different from vector alone (P Յ 0.05). Treatment with diolabeled oligonucleotides. The second band seen with IVP dbcAMP plus LRH-1 was significantly different from LRH-1 alone LRH-1 and probe 2 may be a truncated variant of LHR-1. (P Յ 0.05). Radiolabeled mutated oligonucleotides did not bind to GCT NE (Fig. 9B). stimulated HSD3B2 in a dose-dependent fashion (Fig. 6). This inhibition was maintained in the presence of PKA path- Discussion way agonist (data not shown). The mechanisms controlling the transformation of the pos- tovulatory follicle into a progesterone producing corpus lu- Definition of the cis-elements in the HSD3B2 promoter teum remain poorly understood. Herein, we present data needed for LRH-1 transactivation suggesting that nuclear hormone receptor, LRH-1, may play Deletion analyses of HSD3B2 promoter were performed to an important role in this process. Our data demonstrate a determine the cis-elements used by LRH-1 to enhance its higher level of LRH-1 in the human corpus luteum, com- Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 6025 Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021

FIG. 7. Deletional analysis of the HSD3B2 5Ј-flanking DNA to determine LRH-1 sensitive regions. GCT cells were transiently transfected with luciferase reporter constructs containing serial deletions of HSD3B2 5Ј-flanking DNA. Transfection of reporter constructs was performed with either empty pcDNA3 expression vector (0.5 ␮g/well) or expression vector containing the coding sequence for LRH-1 (0.5 ␮g/well). After recovery for 24 h, cells were lysed, and luciferase activity was measured. Luc on the Y-axis corresponds to empty vector. Results are expressed as a percentage of the empty reporter vector and represent the mean Ϯ SE of data from three to four independent experiments. Ϫ963 and Ϫ345 were significantly different from vector alone (P Յ 0.05).

FIG. 8. Mutational analysis of the HSD3B2 5Ј-flanking DNA to determine LRH-1 sensitive regions. GCT cells were transiently transfected with luciferase reporter constructs containing mutations of the two putative LRH-1 binding sites in the HSD3B2 5Ј-flanking DNA. Transfection of reporter constructs was performed with either empty pcDNA3 expression vector (0.5 ␮g/well) or expression vector containing the coding sequence for LRH-1 (0.5 ␮g/well). After recovery for 24 h, cells were lysed, and luciferase activity was measured. Results are expressed as a percentage of the basal reporter activity of the Ϫ963 and represent the mean Ϯ SE of data from three to four independent experiments. LRH-1 stimulation of mutations 1 and 2 and double mutation are significantly different from LRH-1 stimulation of wild type (P Յ 0.01). pared with mature ovarian follicle and correlation between ovary, LRH-1 has higher expression in preovulatory follicle the expression levels of LRH-1 and HSD3B2 in human corpus and corpus luteum, compared with SF-1. Furthermore, luteum. Clinical treatments with GnRH agonist and recom- LRH-1 was much more highly expressed in granulosa cells binant human gonadotropin before tissue collection proba- than SF-1 (30). bly did not affect the data because expressions of HSD3B2 In macaques undergoing controlled ovarian stimulation, and LRH-1 mRNAs and their positive correlation are similar HSD3B2 mRNA increased within 12 h of HCG administra- in HLGCs, compared with corpus luteum and follicle. In tion (42). In human ovarian cycle, LH surge is essential for addition, we demonstrate that LRH-1 can activate transcrip- ovulation and subsequent conversion of mature ovarian fol- tion of HSD3B2 reporter constructs. licle to corpus luteum. The expression of SF-1 was found to The relatively high expression of LRH-1, compared with be down-regulated in ovarian cells after LH surge (30, 33–35), SF-1, in the human ovarian cortex, mature ovarian follicle, which raises the question regarding the role of SF-1 in corpus and corpus luteum extends a previous report demonstrating luteum steroidogenesis. LRH-1 enhances transcription of high abundance of LRH-1 transcripts in the human ovary by HSD3B2 in a dose-dependent manner and to a greater degree real-time PCR (31). It also agrees with previous studies dem- than SF-1 in human GCT cells, which is consistent with a onstrating a high expression of LRH-1 in mouse and equine previous report using human embryonic kidney-293 cells ovaries by Northern analysis (29, 30). Interestingly, in equine (31). Furthermore, using real-time PCR, we demonstrated a 6026 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2 Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021

FIG. 9. LRH-1 can bind to the two putative cis- elements in the HSD3B2 promoter in EMSA. The two labeled oligonucleotides (FP1 and FP2) were incubated with 5.0 ␮g IVP LRH-1, GCT cell NE (GCT NE), or HLGC nuclear extract (HLGC NE). A, Nonradiolabeled competitor DNA (cold) was added to a 100-fold molar excess to identify nonspecific protein/DNA interaction. B, Radiolabeled mutated DNAs (mutated probes 1 and 2) were used to identify nonspecific protein/DNA interaction. The resulting protein/DNA complexes (shown by arrows) were separated from FP by electrophoresis. Experiments were repeated twice.

high correlation between the expression of HSD3B2 and (24) and inhibits SF-1-mediated transactivation of target LRH-1. This correlation was absent for SF-1. These findings genes. HSD3B2 promoter was shown previously to be in- raise the possibility that LRH-1 rather SF-1 could play an hibited by DAX-1 in the presence or absence of PKA agonist essential role in the transition of ovarian steroidogenesis (44). In the present study, DAX-1 inhibited LRH-1 stimulated from estrogen to progesterone. HSD3B2 in a dose-dependent fashion. This inhibition may be Previous study suggests that stimulation of PKA pathway mediated through direct interaction of LRH-1 with DAX-1 resulted in increased expression of HSD3B2 in ovarian thecal (45). cells (43). Even though dbcAMP increased HSD3B2 tran- Deletion, mutation, and EMSA analyses of the HSD3B2 scription almost 3-fold, the stimulation of HSD3B2 by LRH-1 promoter allowed the identification of two putative LRH-1 was weakly augmented in the presence of dbcAMP. This binding sites that appear to be important regulators for positive response of PKA pathway agonist on LRH-1 stim- HSD3B2 transcription. These two sites are different than ulated HSD3B2 expression raises the possibility that the reg- a previously described SF-1 regulatory element in the ulation of HSD3B2 transcription in ovarian granulosa cells HSD3B2 promoter after study in an adrenal cell model may involve the PKA pathway. (22). This difference may be due to the possibility that both DAX-1 colocalizes with SF-1 during mouse development SF-1 and LRH-1 have slightly different DNA binding spec- Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 6027 ificities or to differences in the adrenal vs. ovarian cell R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302–2312 models. There might also be tissue-specific differences in 15. Wang ZJ, Jeffs B, Ito M, Achermann JC, Yu RN, Hales DB, Jameson JL 2001 the orphan nuclear receptor expression and their mecha- Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1. nisms of control and cis-elements involved in the regula- Proc Natl Acad Sci USA 98:7988–7993 16. Sugawara T, Kiriakidou M, McAllister JM, Kallen CB, Strauss III JF 1997 tion of HSD3B2 gene expression. In addition, the deletion Multiple steroidogenic factor 1 binding elements in the human steroido- analysis suggests that there could be one or more repressor genic acute regulatory protein gene 5Ј-flanking region are required for elements between Ϫ585 to Ϫ345 in the HSD3B2 promoter. maximal promoter activity and cyclic AMP responsiveness. Biochemistry 36:7249–7255 The complete regulation of HSD3B2 promoter will need to 17. Sugawara T, Holt JA, Kiriakidou M, Strauss III, JF1996. Steroidogenic factor be further elucidated. 1-dependent promote activity of the human steroidogenic acute regulatory In conclusion, our findings suggest that LRH-1 is highly protein (StAR) gene. Biochemistry 35:9052–9059 18. Sandhoff TW, Hales DB, Hales KH, McLean MP 1998 Transcriptional reg- expressed in corpus luteum and plays an essential role in ulation of the rat steroidogenic acute regulatory protein gene by steroidogenic the regulation of HSD3B2. Furthermore, we believe that factor 1. Endocrinology 139:4820–4831 Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021 LRH-1 could be the major transcriptional factor respon- 19. Clemens JW, Lala DS, Parker KL, Richards JS 1994 Steroidogenic factor-1 binding and transcriptional activity of the cholesterol side-chain cleavage sible for the shift in human ovarian steroidogenesis from promoter in rat granulosa cells. Endocrinology 134:1499–1508 estrogen predominant to progesterone predominant 20. Chau YM, Crawford PA, Woodson KG, Polish JA, Olson LM, Sadovsky Y Ј Ј milieu. 1997 Role of steroidogenic-factor 1 in basal and 3 ,5-cyclic adenosine monophosphate-mediated regulation of cytochrome P450 side-chain cleavage enzyme in the mouse. Biol Reprod 57:765–771 Acknowledgments 21. Hu MC, Hsu NC, Pai CI, Wang CK, Chung BC 2001 Functions of the upstream and proximal steroidogenic factor 1 (SF-1)-binding sites in the CYP11A1 promoter in basal transcription and hormonal response. Mol Endocrinol 15:812– Received May 20, 2003. Accepted August 26, 2003. 818 Address all correspondence and requests for reprints to: George R. 22. Leers-Sucheta S, Morohashi KI, Mason JI, Melner MH 1997 Synergistic Attia, M.D., Department of Obstetrics and Gynecology, University of activation of the human type II 3␤-hydroxysteroid dehydrogenase/⌬5-⌬4 Miami, 7007 Holtz Center, JMH East Tower, 1611 NW 12th Avenue, isomerase promoter by the transcription factor steroidogenic factor-1/adrenal Miami, Florida 33136. E-mail: [email protected]. 4-binding protein and phorbol ester. J Biol Chem 272:7960–7967 23. Deleted in proof. 24. Ikeda Y, Swain A, Weber TJ, Hentges KE, Zanaria E, Lalli E, Tamai KT, References Sassone-Corsi P, Lovell-Badge R, Camerino G, Parker KL 1996 Steroidogenic factor 1 and Dax-1 colocalize in multiple cell lineages: potential links in en- 1. Suzuki T, Sasano H, Kimura N, Tamura M, Fukaya T, Yajima A, Nagura H docrine development. Mol Endocrinol 10:1261–1272 1994 Immunohistochemical distribution of progesterone, androgen and oes- 25. Becker-Andre M, Andre E, DeLamarter JF 1993 Identification of nuclear trogen receptors in the human ovary during the menstrual cycle: relationship receptor mRNAs by RT-PCR amplification of conserved zinc-finger motif to expression of steroidogenic enzymes. Hum Reprod 9:1589–1595 sequences. Biochem Biophys Res Commun 194:1371–1379 2. Suzuki T, Sasano H, Tamura M, Aoki H, Fukaya T, Yajima A, Nagura H, 26. Galarneau L, Pare JF, Allard D, Hamel D, Levesque L, Tugwood JD, Green Mason JI 1993 Temporal and spatial localization of steroidogenic enzymes in S, Belanger L 1996 The ␣1-fetoprotein locus is activated by a nuclear receptor premenopausal human ovaries: in situ hybridization and immunohistochem- of the Drosophila FTZ-F1 family. Mol Cell Biol 16:3853–3865 ical study. Mol Cell Endocrinol 97:135–143 27. Li M, Xie YH, Kong YY, Wu X, Zhu L, Wang Y 1998 Cloning and char- 3. Doody KJ, Lorence MC, Mason JI, Simpson ER 1990 Expression of messenger acterization of a novel human hepatocyte transcription factor, hB1F, which ribonucleic acid species encoding steroidogenic enzymes in human follicles binds and activates enhancer II of hepatitis B virus. J Biol Chem 273:29022– and corpora lutea throughout the menstrual cycle. J Clin Endocrinol Metab 29031 70:1041–1045 28. Nitta M, Ku S, Brown C, Okamoto AY, Shan B 1999 CPF: an orphan nuclear Sano Y, Suzuki K, Arai K, Okinaga S, Tamaoki BI 4. 1981 Changes in enzyme receptor that regulates liver-specific expression of the human cholesterol 7␣- activities related to steroidogenesis in human ovaries during the menstrual hydroxylase gene. Proc Natl Acad Sci USA 96:6660–6665 cycle. J Clin Endocrinol Metab 52:994–1001 29. Repa JJ, Mangelsdorf DJ 1999 Nuclear receptor regulation of cholesterol and 5. Clyne CD, Speed CJ, Zhou J, Simpson ER 2002 Liver receptor homologue-1 bile acid metabolism. Curr Opin Biotechnol 10:557–563 (LRH-1) regulates expression of aromatase in preadipocytes. J Biol Chem 30. Boerboom D, Pilon N, Behdjani R, Silversides DW, Sirois J 2000 Expression 277:20591–20597 and regulation of transcripts encoding two members of the NR5A nuclear 6. Hanley NA, Rainey WE, Wilson DI, Ball SG, Parker KL 2001 Expression receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and profiles of SF-1, DAX1, and CYP17 in the human fetal adrenal gland: potential NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology interactions in gene regulation. Mol Endocrinol 15:57–68 7. Young M, McPhaul MJ 1998 A steroidogenic factor-1-binding site and cyclic 141:4647–4656 adenosine 3Ј,5Ј-monophosphate response element-like elements are required 31. Sirianni R, Seely JB, Attia G, Stocco DM, Carr BR, Pezzi V, Rainey WE 2002 for the activity of the rat aromatase promoter in rat Leydig tumor cell lines. Liver receptor homologue-1 is expressed in human steroidogenic tissues and Endocrinology 139:5082–5093 activates transcription of genes encoding steroidogenic enzymes. J Endocrinol 8. Morohashi K, Zanger UM, Honda S, Hara M, Waterman MR, Omura T 1993 174:R13–R17 32. Wang ZN, Bassett M, Rainey WE 2001 Liver receptor homologue-1 is ex- Activation of CYP11A and CYP11B gene promoters by the steroidogenic cell- ␤ specific transcription factor, Ad4BP. Mol Endocrinol 7:1196–1204 pressed in the adrenal and can regulate transcription of 11 -hydroxylase. J Mol 9. Sasano H, Mori T, Sasano N, Nagura H, Mason JI 1990 Immunolocalization Endocrinol 27:255–258 of 3 ␤-hydroxysteroid dehydrogenase in human ovary. J Reprod Fertil 89: 33. Agarwal P, Peluso JJ, White BA 1996 Steroidogenic factor-1 expression is 743–751 transiently repressed and c-myc expression and deoxyribonucleic acid syn- 10. McAllister JM, Kerin JF, Trant JM, Estabrook RW, Mason JI, Waterman MR, thesis are induced in rat granulosa cells during the periovulatory period. Biol Simpson ER 1989 Regulation of cholesterol side-chain cleavage and 17 ␣- Reprod 55:1271–1275 hydroxylase/lyase activities in proliferating human theca interna cells in long 34. Fitzpatrick SL, Carlone DL, Robker RL, Richards JS 1997 Expression of term monolayer culture. Endocrinology 125:1959–1966 aromatase in the ovary: down-regulation of mRNA by the ovulatory lutein- 11. Couet J, Martel C, Dupont E, Luu-The V, Sirard MA, Zhao HF, Pelletier G, izing hormone surge. Steroids 62:197–206 Labrie F 1990 Changes in 3 ␤-hydroxysteroid dehydrogenase/␦5-␦4 isomerase 35. Carlone DL, Richards JS 1997 Functional interactions, phosphorylation, and messenger ribonucleic acid, activity and protein levels during the estrous cycle levels of 3Ј,5Ј-cyclic adenosine monophosphate-regulatory element binding in the bovine ovary. Endocrinology 127:2141–2148 protein and steroidogenic factor-1 mediate hormone-regulated and constitu- 12. Martel C, Labrie C, Couet J, Dupont E, Trudel C, Luu-The V, Takahashi M, tive expression of aromatase in gonadal cells. Mol Endocrinol 11:292–304 Pelletier G, Labrie F 1990 Effects of human chorionic gonadotropin (hCG) and 36. Rainey WE, Bird IM, Byrd W, Carr BR 1993 Effect of angiotensin II on human prolactin (PRL) on 3 ␤-hydroxy-5-ene-steroid dehydrogenase/␦5-␦4 isomerase luteinized granulosa cells. Fertil Steril 59:143–147 (3 ␤-HSD) expression and activity in the rat ovary. Mol Cell Endocrinol 72: 37. Freije WA, Pezzi V, Arici A, Carr BR, Rainey WE 1997 Expression of 11 R7–R13 ␤-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the human 13. Parker KL, Schimmer BP 1997 Steroidogenic factor 1: a key determinant of fetal adrenal. J Soc Gynecol Investig 4:305–309 endocrine development and function. Endocr Rev 18:361–377 38. Ramayya MS, Zhou J, Kino T, Segars JH, Bondy CA, Chrousos GP 1997 14. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud Steroidogenic factor 1 messenger ribonucleic acid expression in steroidogenic 6028 J Clin Endocrinol Metab, December 2003, 88(12):6020–6028 Peng et al. • LRH-1 in Regulation of Human Corpus Luteum HSD3B2

and nonsteroidogenic human tissues: Northern blot and in situ hybridization 42. Chaffin CL, Dissen GA, Stouffer RL 2000 Hormonal regulation of steroido- studies. J Clin Endocrinol Metab 82:1799–1806 genic enzyme expression in granulosa cells during the peri-ovulatory interval 39. Zhao L, Bakke M, Krimkevich Y, Cushman LJ, Parlow AF, Camper SA, in monkeys. Mol Hum Reprod 6:11–18 Parker KL 2001 Steroidogenic factor 1 (SF1) is essential for pituitary gonado- 43. McGee EA, Sawetawan C, Bird I, Rainey WE, Carr BR 1996 The effect of trope function. Development 128:147–154 insulin and insulin-like growth factors on the expression of steroidogenic 40. Sawetawan C, Milewich L, Word RA, Carr BR, Rainey WE 1994 Compart- enzymes in a human ovarian thecal-like tumor cell model. Fertil Steril 65:87–93 mentalization of type I 17 ␤-hydroxysteroid oxidoreductase in the human 44. Lalli E, Melner MH, Stocco DM, Sassone-Corsi P 1998 DAX-1 blocks steroid ovary. Mol Cell Endocrinol 99:161–168 production at multiple levels. Endocrinology 139:4237–4243 41. Wang XL, Bassett M, Zhang Y, Yin S, Clyne C, White PC, Rainey WE 2000 45. Suzuki T, Kasahara M, Yoshioka H, Umesono K, Morohashi K 2002 LXXLL Transcriptional regulation of human 11␤-hydroxylase (hCYP11B1). Endocri- motifs in Dax-1 have target specificity for the orphan nuclear receptors nology 141:3587–3594 Ad4BP/SF-1 and LRH-1. Endocr Res 28:537 Downloaded from https://academic.oup.com/jcem/article/88/12/6020/2661519 by guest on 25 September 2021